Effects of Psa and F1 on the adhesive and invasive interactions of Yersinia pestis with human respiratory tract epithelial cells

Yersinia pestis, the causative agent of plague, expresses the Psa fimbriae (pH 6 antigen) in vitro and in vivo. To evaluate the potential virulence properties of Psa for pneumonic plague, an Escherichia coli strain expressing Psa was engineered and shown to adhere to three types of human respiratory tract epithelial cells. Psa binding specificity was confirmed with Psa-coated polystyrene beads and by inhibition assays. Individual Y. pestis cells were found to be able to express the capsular antigen fraction 1 (F1) concomitantly with Psa on their surface when analyzed by flow cytometry. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties of Y. pestis, isogenic Deltacaf (F1 genes), Deltapsa, and Deltacaf Deltapsa mutants were constructed and studied with the three respiratory tract epithelial cells. The Deltapsa mutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the Deltacaf and Deltacaf Deltapsa mutants, indicating that Psa acts as an adhesin for respiratory tract epithelial cells. An antiadhesive effect of F1 was clearly detectable only in the absence of Psa, underlining the dominance of the Psa+ phenotype. Both F1 and Psa inhibited the intracellular uptake of Y. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas Psa seems to block bacterial uptake by interacting with a host receptor that doesn't direct internalization. The Deltacaf Deltapsa double mutant bound and invaded all three epithelial cell types well, revealing the presence of an undefined adhesin(s) and invasin(s).     

Binding of Escherichia coli S fimbriae to human kidney epithelium.

Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium. No binding to connective tissue elements was observed. The results suggest that the biological function of S fimbriae is to mediate the adhesion of E. coli to human epithelial and vascular endothelial cells.     

Two hypolipidemic peroxisome proliferators increase the number of lamellar bodies in alveolar cells type II of the rat lung

Male rats treated with either clofibrate or nafenopin, two peroxisome proliferating compounds with potent hypolipidemic properties, show identical structural changes in their lungs. In both cases, two types of lung cells were affected by these agents: (i) the alveolar epithelial cells type II and (ii) the intraalveolar macrophages. These lung cells are known to be involved in the metabolism of the pulmonary surfactant which serves to reduce the surface tension within alveoli. The size of the alveolar cells type II was conspicuously increased in the treated lungs. Compared to controls, intraalveolar macrophages apparently were slightly more numerous. Their enlarged cytoplasm was strongly vacuolated. The osmiophilic lamellar bodies within alveolar cells type II represent the intracellular presecretory pulmonary surfactant. Their number per individual alveolar cell type II was estimated by means of light microscopic morphometry on Epon-embedded semithin sections. Compared to control lungs, the number was increased by about 30% in rats treated with clofibrate (11.4 +/- 0.5 lamellar inclusions in the control cells; P less than 0.001). The increase in the number of lamellar bodies per type II cell was close to 60% in animals fed the nafenopin diet. In contrast the frequency of alveolar cells type II, estimated per area of lung tissue, remained unchanged. These results demonstrate that clofibrate and nafenopin, two drugs with hypolipidemic properties, cause identical structural changes in the rodent lung. It is concluded from these data that (i) the morphological changes observed in the surfactant metabolizing cells represent a specific action of hypolipidemic agents at the lungs and (ii) hypolipidemic peroxisome proliferators influence the metabolism of the pulmonary surfactant.     

Identification of a cell membrane protein that binds alveolar surfactant.

Alveolar surfactants are complex mixtures of proteins and phospholipids produced by type II alveolar cells and responsible for lowering pulmonary surface tension. The process by which surfactant is produced and exported and by which its production by pulmonary cells is regulated are not well understood. This study was designed to identify a cellular receptor for surfactant constituents. To do so, monoclonal anti-idiotypic antibodies directed against antibodies to porcine and rabbit surfactant proteins were prepared. These monoclonal anti-idiotypic antibodies bind both alveolar lining and bronchial epithelial cells in rabbit, porcine, and human lungs. Macrophages and other nonepithelial cells do not react with these antibodies. Western blot analysis indicates that both A2R and A2C recognize the same proteins in both pig and rabbit lungs: a 30-kd protein and additional proteins at 52 and 60 kd. Preincubating lung wash cells with A2C or A2R prevents binding of porcine or rabbit surfactant preparations, respectively, by these cells. Preincubating frozen sections of lung tissue with surfactant inhibits binding of A2R and A2C to the lung. Antibody directed to a cell membrane protein that recognizes alveolar surfactant may be useful in elucidating the structure and function of this receptor and in understanding the cellular physiology and pathophysiology of the surfactant system.     

IL-33 induces production of autoantibody against autologous respiratory epithelial cells: a potential mechanism for the pathogenesis of COPD

The mechanisms underlying the chronic, progressive airways inflammation, remodelling and alveolar structural damage characteristic of human chronic obstructive pulmonary disease (COPD) remain unclear. In the present study, we address the hypothesis that these changes are at least in part mediated by respiratory epithelial alarmin (IL-33)-induced production of autoantibodies against airways epithelial cells. Mice immunized with homologous, syngeneic lung tissue lysate along with IL-33 administered directly to the respiratory tract or systemically produced IgG autoantibodies binding predominantly to their own alveolar type II epithelial cells, along with increased percentages of Tfh cells and B2 B-cells in their local, mediastinal lymph nodes. Consistent with its specificity for respiratory epithelial cells, this autoimmune inflammation was confined principally to the lung and not other organs such as the liver and kidney. Furthermore, the serum autoantibodies produced by the mice bound not only to murine, but also to human alveolar type II epithelial cells, suggesting specificity for common, cross-species determinants. Finally, concentrations of antibodies against both human and murine alveolar epithelial cells were significantly elevated in the serum of patients with COPD compared with those of control subjects. These data are consistent with the hypothesis that IL-33 contributes to the chronic, progressive airways obstruction, inflammation and alveolar destruction characteristic of phenotypes of COPD/emphysema through induction of autoantibodies against lung tissue, and particularly alveolar type II epithelial cells.     

Alveolar type II cells isolated after silica-induced lung injury in rats have increased surfactant protein A (SP-A) receptor activity.

We examined surfactant secretion and its regulation by surfactant protein A (SP-A) in alveolar type II cells isolated from silica-treated rats to determine the role of SP-A-mediated regulatory control of phospholipid secretion in the pathogenesis of silica-induced alveolar proteinosis. Type II cells were isolated at weekly intervals for 28 d after silica or saline instillation. The maximum total binding of [125I]SP-A (internalized and surface-bound SP-A) to type II cells increased with time after silica instillation and, at 21 d, was 4-fold greater than that of type II cells isolated from saline-treated rats (272.8 +/- 42.5 and 65.4 +/- 9.8 ng/10(5) cells, respectively; P less than 0.05). Type II cells isolated from silica-treated rats showed a 2-fold increased surface binding and a 3-fold increased internalization compared to control cells. The receptor affinity for SP-A was the same for type II cells isolated from silica- and saline-treated animals. Type II cells isolated 14 d after silica instillation were separated into normotrophic and hypertrophic populations by centrifugal elutriation. Hypertrophic cells showed significantly elevated maximum total binding compared to normotrophic cells. The secretion of [3H]phosphatidylcholine [( 3H]PC) by type II cells from silica- and saline-treated animals was also compared. Type II cells from silica-treated animals showed lower basal and tetradecanoyl phorbol acetate (TPA)-stimulated [3H]PC secretion than cells from saline-treated animals at each time point after instillation. SP-A inhibited TPA-stimulated [3H]PC secretion similarly in type II cells isolated after either silica or saline instillation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung

We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.     

Invasion of epithelial cells and proteolysis of cellular focal adhesion components by distinct types of Porphyromonas gingivalis fimbriae

Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nalpha-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration.     

Binding characteristics of Escherichia coli adhesins in human urinary bladder.

We studied domains in the human bladder that acted as receptors for Escherichia coli P, S, type 1, type 1C, and O75X fimbriae or adhesin and domains in the human kidneys that were receptors for E. coli type 1C fimbriae. Binding sites in frozen tissue sections were localized by direct staining with fluorochrome-labeled recombinant strains and by indirect immunofluorescence with the purified adhesins. In the bladder, the P and S fimbriae showed closely similar binding to the epithelial and muscular layers, and the S fimbriae also bound to the connective tissue elements. Type 1 fimbriae bound to vascular walls and to muscle cells, whereas the O75X adhesin bound avidly to connective tissue elements and to some extent to epithelial and muscle cells of the bladder. The type 1C fimbriae bound to distal tubules and collecting ducts of the kidney and to vascular endothelial cells in both the kidney and bladder. The binding of all adhesin types was inhibited by specific receptor analogs or Fab fragments. The results reveal a possible mechanism by which the type 1C fimbriae may help invasion of E. coli in the kidneys but do not support a pathogenetic role for type 1 fimbriae. Similar tissue specificity of P and S fimbriae in the human urinary tract indicates that the presence of binding sites on uroepithelia does not fully explain the virulence properties of P fimbriae in human urinary tract infections.     

Reconstruction of alveolus-like structure from alveolar type II epithelial cells in three-dimensional collagen gel matrix culture.

The purpose of this study is to reconstruct an alveolus-like structure from alveolar type II epithelial cells in a culture condition. Isolated alveolar type II epithelial cells of the rat were cultured in a three-dimensional collagen gel matrix. Single type II cells formed cellular aggregates that had a lumen after cell division in this culture condition. Through proliferation of the component cells, these aggregates grew to assume a globular or branching structure, part of which in turn developed into a large, cystic alveolus-like structure. This structure consisted of flattened epithelial cells intermingled with cuboidal epithelial cells. In these structures, the surfactant production was confirmed by immunohistochemistry and electron microscopy. To our knowledge, this is the first report of a reconstruction of an alveolus-like structure in a three-dimensional collagen gel matrix culture. This culture system seems to provide an appropriate physiological environment in which to study the differentiation and disorders of pulmonary alveoli.     

Platelet activating factor receptor regulates colitis-induced pulmonary inflammation through the NLRP3 inflammasome

Extra-intestinal manifestations (EIM) are common in inflammatory bowel disease (IBD). One such EIM is sub-clinical pulmonary inflammation, which occurs in up to 50% of IBD patients. In animal models of colitis, pulmonary inflammation is driven by neutrophilic infiltrations, primarily in response to the systemic bacteraemia and increased bacterial load in the lungs. Platelet activating factor receptor (PAFR) plays a critical role in regulating pulmonary responses to infection in conditions, such as chronic obstructive pulmonary disease and asthma. We investigated the role of PAFR in pulmonary EIMs of IBD, using dextran sulfate sodium (DSS) and anti-CD40 murine models of colitis. Both models induced neutrophilic inflammation, with increased TNF and IL-1β levels, bacterial load and PAFR protein expression in mouse lungs. Antagonism of PAFR decreased lung neutrophilia, TNF, and IL-1β in an NLRP3 inflammasome-dependent manner. Lipopolysaccharide from phosphorylcholine (ChoP)-positive bacteria induced NLRP3 and caspase-1 proteins in human alveolar epithelial cells, however antagonism of PAFR prevented NLRP3 activation by ChoP. Amoxicillin reduced bacterial populations in the lungs and reduced NLRP3 inflammasome protein levels, but did not reduce PAFR. These data suggest a role for PAFR in microbial pattern recognition and NLRP3 inflammasome signaling in the lung.     

Immunohistochemical study of human pulmonary surfactant apoproteins with monoclonal antibodies. Pathologic application for hyaline membrane disease.

Three monoclonal antibodies, PC6, PE10, and PE12, were used for immunohistochemical studies of human lungs by immunoperoxidase staining. Monoclonal antibodies PC6 and PE10 against pulmonary surfactant apoproteins stained faint granules in the cytoplasm of some alveolar wall cells in adult lung. These stained cells appeared to be alveolar Type II cells. A fetal lung of 20 weeks' gestation had no any positive staining. However, a few scattered positive cells were observed in a newborn lung of 31 weeks' gestation, and the stained cells increased progressively with increasing gestational age. The positively stained cells were very few in the lungs of newborns who died of respiratory distress syndrome (RDS), but the lungs of newborns who died of other causes after recovery from RDS showed many positively stained cells. These results suggest that the immunohistochemical demonstration of the monoclonal antibodies PC6 and PE10 could be a good pathodiagnostic indicator reflecting the localization and development of pulmonary surfactant by alveolar Type II cells. On the other hand, monoclonal antibody PE12 was found to recognize the antigen that occurs on the surfaces of the alveoli of fetal, newborn, and adult lungs as one component of the alveolar lining layer, different from pulmonary surfactant.     

Lysosomal acid lipase over-expression disrupts lamellar body genesis and alveolar structure in the lung

The functional role of neutral lipids in the lung is poorly understood. Lysosomal acid lipase (LAL) is a critical enzyme in hydrolysis of cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Human LAL was over-expressed in a doxycycline-controlled system in mouse respiratory epithelial cells to accelerate intracellular neutral lipid degradation and perturb the surfactant homeostasis in the lung. In this animal system, neutral lipid concentrations of pulmonary surfactant were reduced in bronchoalveolar lavage fluid (BALF) in association with decrease of surfactant protein C (SP-C) gene expression. The size and the number of lamellar bodies in alveolar type II epithelial cells (AT II cells) were significantly reduced accordingly. The number of macrophages required for surfactant recycling in BALF was also significantly reduced. As a result of these combinatory effects, emphysema of the alveolar structure was observed. Taken together, neutral lipid homeostasis is essential for maintenance of lamellar body genesis and the alveolar structure in the lung.     

Hormones and the lung. II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung.

The type II pulmonary epithelial cell is the recognized state of surfactant synthesis and storage. Results of recent studies indicate that the thyroid hormones, triiodothyronine (T3) and thyroxine (T4), may be important regulators of surfactant production and/or release. Direct and indirect immunofluorescence techniques were used in an attempt to demonstrate binding of T3 and T4 in monolayer cultures of isolated type II cells. These cultured epithelial cells are clonally-derived from adult rat lung, retain a diploid karyotype through 35 population doublings in vitro, contain granular inclusions (lamellar bodies) in the perinuclear cytoplasm, and synthesize phosphatidylcholine via the CDP-choline pathway. In isolated type II cells, either of two fluorescent patterns was observed: (a) nuclear fluorescence accompanied by a reticular perinuclear network; or (b) diffuse cytoplasmic accumulations with concentrations around perinculear cytoplasmic inclusions. Ultrastructurally these inclusions had the typical appearance of lamellar bodies. Histochemical studies demonstrated that these inclusions contained surfactant-associated nonspecific esterases and stained with Nile blized markers for pulmonary surfactant indicate that these inclusions are indeed lamellar bodies, the putative sites of surfactant synthesis and/or storage. These findings suggest that the type II pulmonary epithelial cell contains specific binding sites for thyroid hormones, and support the hypothesis that thyroid hormones are regulators of surfactant metabolism.     

The surfactant system of the adult lung: physiology and clinical perspectives

Summary Pulmonary surfactant is synthesized and secreted by alveolar type II cells and constitutes an important component of the alveolar lining fluid. It comprises a unique mixture of phospholipids and surfactant-specific proteins. More than 30 years after its first biochemical characterization, knowledge of the composition and functions of the surfactant complex has grown considerably. Its classically known role is to decrease surface tension in alveolar air spaces to a degree that facilitates adequate ventilation of the peripheral lung. More recently, other important surfactant functions have come into view. Probably most notable among these, surfactant has been demonstrated to enhance local pulmonary defense mechanisms and to modulate immune responses in the alveolar milieu. These findings have prompted interest in the role and the possible alterations of the surfactant system in a variety of lung diseases and in environmental impacts on the lung. However, only a limited number of studies investigating surfactant changes in human lung disease have hitherto been published. Preliminary results suggest that surfactant analyses, e.g., from bronchoalveolar lavage fluids, may reveal quantitative and qualitative abnormalities of the surfactant system in human lung disorders. It is hypothesized that in the future, surfactant studies may become one of our clinical tools to evaluate the activity and severity of peripheral lung diseases. In certain disorders they may also gain diagnostic significance. Further clinical studies will be necessary to investigate the potential therapeutic benefits of surfactant substitution and the usefulness of pharmacologic manipulation of the secretory activity of alveolar type II cells in pulmonary medicine.Abbreviations PC phospatidyleholine - DPPC dipalmitoylphosphatidylcholine - PG phosphatidylglycerol - PI phosphatidylinositol - SP-A, SP-B SP-C, SP-D surfactant-specific proteins A, B, C, and D - ARDS adult respiratory distress syndrome - IRDS infant respiratory distress syndrome - BAL bronchoalveolar lavage - IPF idiopathic pulmonary fibrosis - HP hypersensitivity pneumonitis - DIPD drug-induced pulmonary disease     

Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization.     

The distribution of osmiophilic lamellae within the alveolar and bronchiolar walls of the mammalian lungs as revealed by osmium-ethanol treatment

Osmiophilia of the cellular elements of the alveoli and bronchioles was investigated using the new technique of second osmication with a solution containing a mixture of osmium tetroxide and ethanol. Previously this method was used to reveal osmiophilic periodic lamellae of synthetic dipalmitoyl lecithin and the surface film of the acellular alveolar lining layer of the mammalian lung in thin sections. In the present experiments, much of the osmiophilic content of inclusions in type II epithelial cells became visible only after second osmication with osmium-ethanol but was not visible with the initial osmium tetroxide fixation. The osmiophilic content of the inclusions showed 42 Å periodicity. These results are in accord with those obtained in the study of dipalmitoyl lecithin and the surface film, suggesting that the inclusions are the precursors of the pulmonary surfactant. Several new observations were made with this technique. These include osmiophilic lamellae within Golgi saccules, within the multivesicular bodies of type II cells, in the vesicles of type I epithelial cells, and in the basement membranes of epithelial cells. These observations are consistent with the idea that the Golgi apparatus and multivesicular bodies are closely related to the production of surfactant and that the surfactant may be disposed of by type I epithelial cells after engulfment by pinocytosis. Thereafter, the surfactant may eventually be cleared through the lymphatics. Clara cell granules behaved differently and were thought to be an unlikely site for the synthesis of the pulmonary surfactant.     

Experimental pulmonary fibrosis induced by trisodium citrate and acid-citrate-dextrose

A single intrapulmonary injection of 3.8% trisodium citrate and acid-citrate-dextrose (ACD) into rabbits results in extensive degeneration and necrosis of alveolar pneumocytes, including the type II pneumocyte, and of bronchiolar or bronchial epithelial cells. Subsequently, the alveoli and alveolar ducts collapse, and the septa and ductal walls adhere to each other, accompanied by the proliferation of interstitial fibroblasts. These fibroblasts produce fibrous connective tissue which is followed by pulmonary fibrosis in 1 week. Epithelial regeneration, especially that resulting from the proliferation of immature type II pneumocytes, occurs around the periphery of the fibrous lesions. The synthesis and release of large amounts of surfactant materials by the proliferated type II pneumocytes may induce the surfactant materials to reopen the air spaces of the collapsed and adhesive alveoli. By 4 weeks those fibrous areas in the pathological lungs become smaller and/or appear normal. These results suggest that this is a useful experimental animal model for pulmonary fibrosis, and that epithelial cells, especially type II pneumocytes, are associated with both the induction of and the recovery from the disorder; in the early stage, interference by reepithelization resulting from type II pneumocyte proliferation may elicit the proliferation of fibroblasts, and in later stages, reepithelization and surfactant synthesis by newly proliferated type II pneumocytes may permit the reopening of collapsed and adhesive air spaces.     

Hormones and the lung II. Immunohistochemical localization of thyroid hormone binding in type II pulmonary epithelial cells clonally-derived from adult rat lung

The type II pulmonary epithelial cell is the recognized site of surfactant synthesis and storage. Results of recent studies indicate that the thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄), may be important regulators of surfactant production and/or release. Direct and indirect immunofluorescence techniques were used in an attempt to demonstrate binding of T³ and T⁴ in monolayer cultures of isolated type II cells. These cultured epithelial cells are clonally-derived from adult rat lung, retain a diploid karyotype through 35 population doublings in vitro, contain granular inclusions (lamellar bodies) in the perinuclear cytoplasm, and synthesize phosphatidylcholine via the CDP-choline pathway. In isolated type II cells, either of two fluorescent patterns was observed: (a) nuclear fluorescence accompanied by a reticular perinuclear network; or (b) diffuse cytoplasmic accumulations with concentrations around perinuclear cytoplasmic inclusions. Ultrastructurally these inclusions had the typical appearance of lamellar bodies. Histochemical studies demonstratedthat these inclusions contained surfactant-associated nonspecific esterases and stained with Nile blue hydrochloride. The positive reactions with these two recognized markers for pulmonary surfactant indicate that these inclusions are indeed lamellar bodies, the putative sites of surfactant synthesis and/or storage. These findings suggest that the type II pulmonary epithelial cell contains specific binding sites for thyroid hormones, and support the hypothesis that thyroid hormones are regulators of surfactant metabolism.     

Immunoultrastructural study of surfactant system. Distribution of specific protein of surface active material in rabbit lung.

Ducks were immunized with rabbit pulmonary surface active material that had been prepared with Sephadex chromatographic separation on lung washings. Specific antibody against a protein of surface active material was obtained after absorption of duck antisera with rabbit serum. Using the ultrastructural immunoperoxidase method, dense reaction product was recognized in alveolar lining material as granular deposits, closely associating with tubular myelin figures that were often observed in the basal layer of alveolar lining layer. In type I pneumocytes, reaction product was noted only in some pinocytotic vesicles. By contrast, in type II pneumocytes there was positivity in association with lamellar inclusion bodies, multivesicular bodies, Golgi complex, and endoplasmic reticulum. No reaction product was detected in bronchiolar and bronchial epithelial cells, including nonciliated bronchiolar and goblet cells. These findings indicate that specific protein in surfactant is synthesized in type II pneumocytes and secreted into the alveolar space to become a part of the alveolar lining layer, thus suggesting that specific protein plays an important role in surfactant system. Furthermore, reaction product could be detected in phagocytic vacuoles in alveolar macrophages and thus seems to be related to the clearance mechanism of the protein component of surfactant.